EP3891286A1 - Récepteurs de surface cellulaire régulables et compositions et procédés associés - Google Patents

Récepteurs de surface cellulaire régulables et compositions et procédés associés

Info

Publication number
EP3891286A1
EP3891286A1 EP19892943.2A EP19892943A EP3891286A1 EP 3891286 A1 EP3891286 A1 EP 3891286A1 EP 19892943 A EP19892943 A EP 19892943A EP 3891286 A1 EP3891286 A1 EP 3891286A1
Authority
EP
European Patent Office
Prior art keywords
cell
protease
domain
surface receptor
cell surface
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19892943.2A
Other languages
German (de)
English (en)
Other versions
EP3891286A4 (fr
Inventor
Crystal Mackall
Louai LABANIEH
Robbie MAJZNER
Michael Z. Lin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
Original Assignee
Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leland Stanford Junior University filed Critical Leland Stanford Junior University
Publication of EP3891286A1 publication Critical patent/EP3891286A1/fr
Publication of EP3891286A4 publication Critical patent/EP3891286A4/fr
Pending legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24232—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24271—Demonstrated in vivo effect

Definitions

  • FIG. 10 A series of images at various time points of the CAR T cells described for FIG. 6 co-cultured with RFP-labeled 143B osteosarcoma cells expressing the B7H3 antigen.
  • the cells were co-cultured in the presence of an inhibitor of the protease.
  • the antigen binding portion of the CAR binds to B7H3, and the CAR includes a 4-1 BB intracellular signaling domain and a O ⁇ 3z intracellular signaling domain. Expression was determined by staining with B7H3 Fc in the presence or absence of an inhibitor of the protease for various cleavage sites.
  • Panel B A graph showing the quantification of the flow cytometry plots of panel A.
  • Panel C A table showing the amino acid sequences and biochemical parameters of the various cleavage sites used.
  • FIG. 31 Data demonstrating that B7H3 SNIP CAR-T cells have greater anti-tumor efficacy compared to constitutive B7H3 CAR-T cells in a medulloblastoma flank model.
  • Panel A A series of bioluminescent images tracking the growth of medulloblastoma cells (MED8A), which express firefly luciferase, and then treated with the indicated CAR-T cells.
  • Panel B Quantification of the bioluminescent imaging of Panel A.
  • FIG. 33 Data demonstrating that CD19 SNIP CAR-T cells have equivalent anti-tumor efficacy compared to constitutive CD19 CAR-T cells in an orthotopic leukemia model.
  • Panel A A series of bioluminescent images tracking the growth of Nalm6 leukemia cells, which express firefly luciferase.
  • Panel B Quantification of the bioluminescent imaging seen in Panel A.
  • cell surface receptors that include an extracellular binding domain, a transmembrane domain, an intracellular signaling domain, and a protease cleavage site disposed between the extracellular binding domain and the intracellular signaling domain.
  • the cell surface receptors are engineered cell surface receptors, such as chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • cells that include such receptors (e.g., where the cells express the receptors on their surface) and pharmaceutical compositions including such cells.
  • Nucleic acids that encode the cell surface receptors, cells including such nucleic acids, and pharmaceutical compositions including such cells are also provided.
  • methods for regulating signaling of a cell surface receptor and methods of using the cells of the present disclosure, including methods of using such cells to administer a regulatable cell-based therapy to an individual.
  • the protease may be expressed in the cell as a molecule separate from the receptor (sometimes referred to herein as a“trans” configuration) or the cell surface receptor molecule itself may further include the protease (sometimes referred to herein as a “cis” configuration).
  • the activity of the protease is regulatable depending upon the presence or absence of an inhibitor of the protease. In the presence of a protease inhibitor, the protease is prevented from cleaving the protease cleavage site, leaving the cell surface receptor intact and capable of transducing ligand (e.g., antigen) binding-related signals into the cell.
  • the ligand comprises an antigen or an antigen-MHC complex.
  • the receptor may include a chain that includes a transmembrane domain and an intracellular signaling domain, and the protease cleavage site may be provided in such a chain between the transmembrane domain and the intracellular signaling domain.
  • Non-limiting examples of engineered receptors include chimeric receptors (e.g., chimeric antigen receptors (CARs)), engineered T cell receptors (TCRs) (e.g., having altered (or“engineered”) specificity and/or affinity for an antigen as compared to a counterpart wild-type TCR, having one or more chains covalently or non-covalently bound (e.g., fused) to one another, and/or the like), chimeric cytokine receptors (CCRs), synthetic notch receptors (synNotch), and the like.
  • CARs chimeric antigen receptors
  • TCRs engineered T cell receptors
  • CCRs e.g., having altered (or“engineered”) specificity and/or affinity for an antigen as compared to a counterpart wild-type TCR, having one or more chains covalently or non-covalently bound (e.g., fused) to one another, and/or the like
  • CCRs chimeric cyto
  • a cell surface receptor of the present disclosure does have a native/wild-type counterpart, and the cell surface receptor differs from the native/wild-type counterpart by virtue of a protease cleavage site (and optionally, a corresponding protease) being recombinantly disposed within the receptor at a selected extracellular or intracellular position, e.g., between the ligand binding domain and the transmembrane domain, between the transmembrane domain and an intracellular signaling domain, etc.
  • a protease cleavage site and optionally, a corresponding protease
  • Non-limiting examples of recombinant cell surface receptors of the present disclosure having native/wild-type counterparts and modified to include a protease cleavage site include stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, immune receptors such as CD28, CD80, ICOS, CTLA4, PD1 , PD-L1 , BTLA, HVEM, CD27, 4-1 BB, 4-1 BBL, 0X40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1 , TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1 , B7- 1 , B7-H1 , and B7-H3, a type I cytokine receptor such as lnterleukin-1 receptor, lnterleukin-2 receptor, ln
  • such a receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.
  • FIG. 4 is a trans SNIP system that includes a CAR having the cleavage site disposed between the extracellular binding domain and the transmembrane domain, and an extracellularly-tethered protease capable of cleaving the cleavage site in the absence of an inhibitor of the protease.
  • FIG. 5 Shown in FIG. 5 is a cis SNIP system that includes a CAR having the cleavage site disposed between the extracellular binding domain and the transmembrane domain, where the CAR further includes the protease disposed between the cleavage site and the transmembrane domain, the protease being capable of cleaving the cleavage site in the absence of an inhibitor of the protease.
  • the CAR includes a costimulatory domain (in these examples, a 4- 1 BB costimulatory domain) C-terminal to the transmembrane domain, and a primary signaling domain (in these examples, a O ⁇ 3z primary signaling domain) C-terminal to the costimulatory domain.
  • a costimulatory domain in these examples, a 4- 1 BB costimulatory domain
  • a primary signaling domain in these examples, a O ⁇ 3z primary signaling domain
  • FIG. 1 Shown in FIG. 1 is a trans SNIP system in which the protease is provided as a separate molecule and intracellularly tethered to the cell membrane.
  • FIG. 3 shows a corresponding cis SNIP system where the CAR further includes the protease disposed between the cleavage site and the intracellular signaling domain nearest to the transmembrane domain.
  • the protease cleavage site is disposed between two of the two or more intracellular signaling domains.
  • An example of such a CAR/SNIP system is schematically illustrated in FIG.
  • the CAR includes a first intracellular signaling domain (here, a O ⁇ 3z primary signaling domain) and a second intracellular signaling domain (here, a 4-1 BB costimulatory domain).
  • Shown in FIG. 2 is a trans SNIP system in which the protease cleavage site is disposed between the first and second intracellular signaling domains, and the protease is provided as a separate molecule and intracellularly tethered to the cell membrane, where the protease is capable of cleaving the cleavage site in the absence of an inhibitor of the protease.
  • a TCR of the present disclosure includes the cleavage site and the protease (cis configuration), e.g., present within a linker.
  • the protease when the cell surface receptor is a TCR, the protease is supplied in trans - that is, not part of the polypeptide chain that includes the cleavage site.
  • the protease when the protease is supplied in trans, the protease is tethered to a different chain of the TCR.
  • the protease when the cleavage site is disposed within the a chain, the protease may be supplied on the b chain, and vice versa.
  • the cleavage site may be disposed within one of the CD3 chains (epsilon, gamma, delta, or zeta), and the protease may be supplied in a different CD3 chain.
  • the extracellular binding domain of the cell surface receptor (e.g., an engineered cell surface receptor such as a CAR, engineered TCR, or the like) specifically binds a molecule on the surface of a target cell.
  • the target cell may be any cell type of interest.
  • the target cell may be a genetically and/or phenotypically normal cell.
  • the target cell is a genetically and/or phenotypically abnormal cell.
  • antibodies which may be employed in the cell surface receptor (e.g., a CAR) of the present disclosure include Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, lcrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Rad
  • variable refers to an antibody that binds to a particular cognate antigen (e.g., HER2 for trastuzumab) but has fewer or more amino acids than the parental antibody, has one or more amino acid substitutions relative to the parental antibody, is a single-chain variant (such as an scFv variant) of the parental antibody, or any combination thereof.
  • a particular cognate antigen e.g., HER2 for trastuzumab
  • a single-chain variant such as an scFv variant
  • Domains that may be employed to tether the protease to the cell membrane include, but are not limited to, domains that are post-translationally modified with one or more moieties that tether proteins to cell membranes in nature, such as domains found in prenylated proteins, fatty acylated proteins, glycosylphosphatidylinositol-linked proteins (GPI), N-myristoylation, S-palmitoylation, and the like.
  • domains that are post-translationally modified with one or more moieties that tether proteins to cell membranes in nature such as domains found in prenylated proteins, fatty acylated proteins, glycosylphosphatidylinositol-linked proteins (GPI), N-myristoylation, S-palmitoylation, and the like.
  • GPI glycosylphosphatidylinositol-linked proteins
  • Polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection.
  • Table 1 Shown in Table 1 below are amino acid sequences of example cell surface receptors and proteases of the present disclosure (from N- to C-terminus), and nucleotide sequences of nucleic acids encoding such cell surface receptors and proteases. Included in these examples are the CARs and proteases employed in the Experimental section below. Not shown are signal sequences initially present at the N-termini of the polypeptides. Segments/domains of the polypeptides are indicated by alternating stretches of underlined and non-underlined text, and the identities of the segments/domains are provided in the left column.
  • cells that include any of the cell surface receptors, proteases, nucleic acids, and/or expression vectors of the present disclosure.
  • cells where a cell surface receptor and/or a protease of the present disclosure is expressed on the surface of the cell By“expressed on the surface of the cell” is meant the cell surface receptor and/or a protease has been trafficked to the cell membrane such that - in the case of a cell surface receptor - the extracellular binding domain is displayed on the cell surface, the transmembrane portion passes through the cell membrane, and the one or more intracellular signaling domains are disposed adjacent to the intracellular side of the cell membrane.
  • T cells include naive T cells (TN), cytotoxic T cells (TCTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), tissue resident memory T cells (TRM), effector T cells (TEFF), regulatory T cells (TREG S ), helper T cells (TH, TH1 , TH2, TH1 7) CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha beta T cells (T ab ), and gamma delta T cells (T Ud ).
  • the cell is a T cell and the cell surface receptor is a CAR, e.g., any of the CARs described herein.
  • transfecting or transducing cells with a nucleic acid or expression vector of the present disclosure.
  • the term“transfection” or“transduction” is used to refer to the introduction of foreign DNA into a cell.
  • a cell has been“transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3 rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw- Hill, and Chu et al. (1981 ) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material.
  • the T cells are transduced with a retroviral vector, e.g., a gamma retroviral vector or a lentiviral vector, encoding the CAR. In some embodiments, the T cells are transduced with a lentiviral vector encoding the CAR.
  • a retroviral vector e.g., a gamma retroviral vector or a lentiviral vector
  • the T cells are transduced with a lentiviral vector encoding the CAR.
  • a specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques.
  • a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38 is further isolated by positive or negative selection techniques.
  • the manufactured T cell compositions do not express one or more of the following markers: CD57, CD244, CD 160, PD-1 , CTLA4, TIM3, and LAG3.
  • the manufactured T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1 , CTLA4, TIM3, and LAG3.
  • T cells are activated and expanded for about 1 to 21 days, e.g., about 5 to 21 days. In some embodiments, T cells are activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of a nucleic acid (e.g., expression vector) encoding the polypeptide into the T cells.
  • a nucleic acid e.g., expression vector
  • cell culture media include, but are not limited to RPMI 1640, Clicks, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • the nucleic acid (e.g., an expression vector) encoding the cell surface receptor is introduced into the cell (e.g., a T cell) by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran- mediated transfer, and the like.
  • the nucleic acid (e.g., expression vector) encoding the cell surface receptor is introduced into the cell (e.g., a T cell) by AAV transduction.
  • viruses that include any of the cell surface receptors, nucleic acids, and/or expression vectors of the present disclosure.
  • compositions comprising any of the cell surface receptors, proteases, nucleic acids, expression vectors, and/or cells described herein.
  • One or more additives such as a salt (e.g., NaCI, MgCI 2 , KCI, MgS0 ), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.
  • a salt
  • compositions are also provided.
  • the pharmaceutical compositions may include any of the cells of the present disclosure, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions generally include a therapeutically effective amount of the cells.
  • therapeutically effective amount is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control.
  • An effective amount can be administered in one or more administrations.
  • the cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents.
  • Formulations of the cells suitable for administration to a patient are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.
  • the cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.
  • parenteral e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.
  • An aqueous formulation of the cell surface receptors, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the formulation to modulate the tonicity of the formulation.
  • Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • the term“isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
  • a surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • the pharmaceutical composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • a pharmaceutical composition that includes a therapeutically effective amount of cells (e.g., T cells, such as CAR T cells) of the present disclosure.
  • A“therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects.
  • the term“therapeutically effective amount” includes an amount that is effective to“treat” an individual (e.g., a patient).
  • a pharmaceutical composition of the present disclosure includes from 1 x10 6 to 5x10 10 of the cells of the present disclosure.
  • provided herein are methods that employ the cell surface receptors, proteases, nucleic acids, expression vectors, and/or cells described herein.
  • methods for regulating signaling of a cell surface receptor include contacting a cell of the present disclosure with an inhibitor of the protease when signaling through the cell surface receptor is desired.
  • the protease is derived from HCV NS3, and the inhibitor of the protease is selected from imeprevir, danoprevir, asunaprevir, grazoprevir, simeprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, and any combination thereof.
  • a regulatable cell-based therapy e.g., CAR cell-based therapy
  • the individual in need thereof has cancer
  • the cell surface receptor e.g., CAR
  • the contacting may include administering to the individual by a suitable route of administration simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, or any combination thereof, in an amount effective to inhibit the protease expressed by the administered cells or progeny thereof.
  • the inhibitor of the protease may be administered to the individual prior to, concurrently with (that is, co-administered), and/or subsequent to administration of the pharmaceutical composition to the individual.
  • the methods may include withholding or ceasing administration of the protease inhibitor to delay or prevent cell exhaustion resulting from CAR activity.
  • T cell exhaustion resulting from CAR activity may be due to antigen-independent tonic signaling and/or prolonged antigen-dependent signaling through antigen engagement.
  • signaling through a CAR may no longer be desired because of adverse side effects caused by the cells or progeny thereof, such that the methods may include ceasing administration of the protease inhibitor to reduce adverse side effects caused by the cells or progeny thereof.
  • Adverse side effects may include, but are not limited to, off tumor effects, toxicity resulting from, e.g., unrestricted antigen-driven proliferation of the cells, and the like. Such toxicity may include cytokine release syndrome and/or neurotoxicity.
  • the methods may further include ceasing administration of the protease inhibitor to reduce adverse side effects caused by the cells or progeny thereof.
  • the amount of signaling through the receptor is tuned by selecting a protease cleavage site having a particular“strength” (where a“stronger” cleavage site is cleaved by the protease more efficiently than a“weaker” cleavage site is cleaved by the protease), the amount of the protease inhibitor administered to the individual, or a combination thereof.
  • a protease cleavage site having a particular“strength” where a“stronger” cleavage site is cleaved by the protease more efficiently than a“weaker” cleavage site is cleaved by the protease
  • the amount of the protease inhibitor administered to the individual or a combination thereof.
  • kits that include any of the nucleic acids and/or expression vectors of the present disclosure, and instructions for introducing the nucleic acid or expression vector into a cell.
  • the expression vector encodes a protease, where the protease cleavage site is a cleavage site for the protease.
  • the expression vector is configured to express the cell surface receptor and the protease from the same promoter.
  • the expression vector may be a bicistronic expression vector for expression of separate cell surface receptor and protease molecules under the same promoter.
  • kits find use in a variety of in vitro, ex vivo, and in vivo applications.
  • the instructions of such kits may further include instructions for regulatable signaling through the cell surface receptor.
  • the instructions of such kits may further include instructions for contacting the cell or progeny thereof with an inhibitor of the protease when signaling through the cell surface receptor is desired.
  • the instructions of such kits may further include instructions for withholding the protease inhibitor and/or ceasing contacting of the inhibitor when signaling through the cell surface receptor is not/no longer desired.
  • kits of the present disclosure may further include any other reagents useful for regulatable signaling of the cell surface receptor, such as transfection/transduction reagents useful for introducing the nucleic acid or expression vector into a cell of interest, e.g., a T cell or other cell of interest.
  • transfection/transduction reagents useful for introducing the nucleic acid or expression vector into a cell of interest, e.g., a T cell or other cell of interest.
  • An engineered cell surface receptor comprising:
  • cell surface receptor of embodiment 1 wherein the cell surface receptor is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the cell surface receptor of embodiment 1 1 wherein the antigen on the surface of the cancer cell is selected from the group consisting of: B7-H3 (CD276), CD19, GD2, CD22, and HER2.
  • TCR T cell receptor
  • the viral protease cleavage site is selected from the group consisting of: an NS4A/4B junction cleavage site, an NS3/NS4A junction cleavage site, an NS4A/NS4B junction cleavage site, an NS4B/NS5A junction cleavage site, an NS5A/NS5B junction cleavage site, and variants thereof cleavable by the viral protease.
  • a cell comprising the cell surface receptor of any one of embodiments 1 to 32.
  • stem cell is selected from the group consisting of: a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), and a neural stem cell (NSC).
  • HSC hematopoietic stem cell
  • iPSC induced pluripotent stem cell
  • MSC mesenchymal stem cell
  • NSC neural stem cell
  • the activity of the protease is regulated depending upon the presence or absence of an inhibitor of the protease.
  • the protease In the presence of a protease inhibitor, the protease is prevented from cleaving the protease cleavage site, leaving the cell surface receptor intact and capable of transducing ligand/antigen binding-related signals to the cell.
  • the protease In the absence of a protease inhibitor, the protease cleaves the protease cleavage site, thereby separating one or more domains of the cell surface receptor from the remaining domains of the cell surface receptor, rendering the cell surface receptor incapable of transducing ligand/antigen binding-related signals to the cell or incapable of binding to ligand/antigen at all.
  • the cell surface receptors are engineered cell surface receptors - in particular, chimeric antigen receptors (CARs).
  • Example 1 A regulatable cell surface receptor having a protease cleavage site disposed between the transmembrane domain and an intracellular signaling domain
  • FIG. 6 panel A is an anti-CD3 z western blot of protein extracted from primary human T cells expressing the CAR and cultured in the presence (+) or absence (-) of 3 m M of the HCV NS3 protease inhibitor grazoprevir.
  • FIG. 6 (panel B) is a flow cytometry plot showing the CAR T cells stained with B7H3-Fc, which recognizes both cleaved and full length surface CAR.
  • FIG. 6 (panel C) is a flow cytometry plot showing the expression of the activation and exhaustion marker LAG-3 on the CAR T cells.
  • FIG. 21 is a graph showing the expression of B7H3 antigen on several tumor cell lines (Nalm6 Med, D425, Nalm6 Hi), as determined by flow cytometric analysis of MGA271 stained cells.
  • FIG. 22 shows a series of graphs showing the GFP fluorescence of the GFP-labeled tumor cells of FIG. 21 .
  • the tumor cells were co-cultured with B7H3-SNIP-BBz T cells in the absence of protease inhibitor (“OFF”).
  • the basal cytotoxic capacity of the B7H3-SNIP-BBz cells in the OFF condition is modulated by the strength of the cleavage site and antigen expression level on the tumor cells.
  • FIG. 27 Shown in FIG. 27 is an 3h ⁇ -0 ⁇ 3z western blot of protein extracted from primary human T cells transduced with a B7H3-SNIP-BBz CAR and cultured in absence of drug to determine the efficiency of proteolytic cleavage of the CAR by the NS3 protease.
  • FIG. 28 Shown in FIG. 28, panel A, is a series of graphs showing the GFP fluorescence of GFP- labeled Nalm6 leukemia cells (left) and 143B osteosarcoma cells (right). As shown, the cytotoxic capacity of B7H3-SNIP-BBz cells can be controlled by the addition of drug, as determined by tumor GFP fluorescence. Mock untransduced T cells served as a negative control. B7H3-CAR T cells lacking the protease cleavage site served as a positive control.
  • FIG. 29 Shown in FIG. 29, panel A, is a series of graphs showing the levels of IFNy and IL-2 cytokine levels in tumor co-cultures after pre-incubation with 3mM grazoprevir (drug) for various amount of time prior to co-culture. Drug was also present during the co-culture. Co-culture supernatant was harvested 24 hours after initiation of the co-culture and analyzed for cytokine secretion by ELISA. Shown in FIG.
  • mice in the“CAR ON” group were also implanted with an osmotic drug pump (Azlet model 2002) containing 54 mg/mL grazoprevir and 0.6 mg/mL ritonavir. These mice were additionally dosed with 50 mg/kg grazoprevir and 25 mg/kg ritonavir by oral gavage 1 -2 times per day. Shown in FIG. 30, panel B, is the quantification of the bioluminescent imaging seen in panel A. As can be seen in the data, GD2-SNIP-BBz CAR-T cells have greater anti-tumor activity in the CAR ON state compared with the standard constitutive CAR (GD2-BBz).
  • mice in the“CAR ON” group were also given 50 mg/kg grazoprevir and 25 mg/kg ritonavir by oral gavage 3 times per day. Shown in panel B is the quantification of the bioluminescent imaging seen in panel A. As can be seen in the data, B7H3- SNIP-BBz CAR-T cells have greater anti-tumor activity in the CAR ON state compared with the standard constitutive CAR (B7H3-BBz).
  • FIG. 32 Data for HER2-SNIP-BBz CAR-T cells and the corresponding constitutive CAR-T cells in an orthotopic osteosarcoma model is provided in FIG. 32. Shown in FIG. 32 is a graph of the tumor size of mice that were implanted with 63.3 osteosarcoma cells, which express firefly luciferase, and then treated with the indicated CAR-T cells. At day 0, NSG mice were infused with 1 x10 6 63.3 cells in the right leg. At day 18 days, 1 x10 7 CAR-T or control cells were implanted by tail vein injection.
  • the improved anti-tumor activity of the SNIP CAR-T cells could be attributed to reduced levels of tonic signaling and dynamic full-length CAR expression profile, which follows the pharmacokinetics of the administered drug.
  • constitutive expression of the corresponding CAR results in high tonic signaling and over-activation of T cells, which might lead to exhaustion and diminished anti-tumor activity.
  • Example 10 - SNIP CAR-T cells display a more functional phenotype, have greater proportions of memory T cells, exhibit reduced expression of exhaustion markers, and are more responsive to ex vivo stimulation than the corresponding constitutive CAR-T cells
  • FIG. 37 Shown in FIG. 37, panel B, is a series of graphs showing plasma cytokine levels of the proinflammatory cytokines IL-2 and IFNy in mice treated with the indicated amount of drug.
  • the data demonstrate that plasma cytokine levels of mice that received CD19-SNIP-BBz CAR-T cells can be tuned by administration of selected doses of the HCV protease inhibitor grazoprevir.
  • plasma cytokine levels of IL-2 and IFNy are low in the absence of drug, indicating that the CAR-T cells are completely“OFF” and do not have observable leaky activity. This is a desirable property of a regulatable CAR-T system which is to be used as a safety switch in clinical applications.
  • Example 12 ROR1-SNIP CAR-T cells extend survival as compared to constitutive ROR1 CAR-T cells in an on-target off-tumor toxicity model
  • ROR1 -SNIP-BBz CAR-T cells were tested in an on-target, off-tumor toxicity model (see Srivastava et al. Cancer Cell 2019).
  • the CAR construct included the anti- ROR1 scFv designated as“clone F” in International Patent Application Publication No. WO 2019/008378.
  • NSG mice were irradiated (2.2 Gy) to initiate toxicity and then 5 hours later treated with 6M ROR1 -SNIP CAR-T cells or constitutive control CAR-T cells. Mice weight was measured every two days.
  • FIG. 38, panel A is a graph showing percent weight change.

Abstract

L'invention concerne des récepteurs de surface cellulaire qui comprennent un domaine de liaison extracellulaire, un domaine transmembranaire, un domaine de signalisation intracellulaire et un site de clivage par les protéases disposé entre le domaine de liaison extracellulaire et le domaine de signalisation intracellulaire. Dans certains aspects, les récepteurs de surface cellulaire sont des récepteurs de surface cellulaire modifiés, tels que des récepteurs d'antigènes chimériques (CAR). L'invention concerne également des cellules comprenant de tels récepteurs (par exemple, des cellules exprimant les récepteurs sur leur surface) et des compositions pharmaceutiques comprenant de telles cellules. L'invention concerne également des acides nucléiques codant pour les récepteurs de surface cellulaire, des cellules comprenant de tels acides nucléiques, et des compositions pharmaceutiques comprenant de telles cellules. L'invention concerne en outre des procédés de régulation d'un récepteur de surface cellulaire, et des procédés d'utilisation des cellules de la présente invention, notamment des procédés d'utilisation de telles cellules pour administrer une thérapie à base de cellules régulables à un individu.
EP19892943.2A 2018-12-06 2019-12-05 Récepteurs de surface cellulaire régulables et compositions et procédés associés Pending EP3891286A4 (fr)

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